Method for obtaining an isolated extract of the plant cyclamen europaeum L. and its use as a therapeutic agent

ABSTRACT

The invention relates to a procedure for obtaining an isolated extract of the plant  Cyclamen europaeum L.  and to its use as a therapeutic agent. In particular, it relates to the use of the isolated extract for preparing a medicament for treating sinusitis. This invention also relates to a method for extracting the isolated extract by aqueous extration and by alcoholic extraction.

FIELD OF THE INVENTION

This invention relates to a procedure for obtaining an isolated extractof the plant Cyclamen europaeum L. and to its use as a therapeuticagent. In particular, it relates to the use of said isolated extract forpreparing a medicament for treating sinusitis.

This invention also relates to a method for extracting said isolatedextract by aqueous extraction and by alcoholic extraction.

BACKGROUND OF THE INVENTION

Known in the state of the art are various methods for treatinginflammation of the mucous membrane or of the paranasal sinuses, inparticular sinusitis. However, current procedures for treatingsinusitis, both acute and chronic, centre above all on three basicproblems to which a solution is to a greater or lesser degree decisiveas regards the efficacy and duration of the treatment. These problemsare: re-establishing ventilation and normal draining of the paranasalsinuses, halting the process of inflammation and swelling of the mucousmembrane and combating the infectious microflora.

Said illnesses are often treated in a conservative way, involving theuse of antimicrobic products which are not always effective, since insome cases they stimulate resistance to the pathogenic microflora ofthese products which, in their turn, are not sufficiently innocuous.

In the first phases of acute sinusitis these treatment proceduresprovide for oral administration of first-line antibiotics such asampicillin and amoxicillin. If these prove to be ineffective, thenamoxicillin and clavulanic acid or cephalosporins of generations II andIII are prescribed [Ellen R. Wald, MD, Chronic sinusitis in children.Journal of Pediatrics, 1995, 127 (3) 339; Inexpensive Antibiotics are asEffective as Newer, More Expensive Ones in Treating Acute BacterialSinusitis. Press Release, Mar. 23, 1999. Agency for Health Care Policyand Research, Rockville, Md.; and Glenn Isaacson, MD, FAAP, FACS,Sinusitis in Childhood, Ped. Clin. of N Am., 1996, 43(6):1305].

The role of antibiotics in treatments for chronic sinusitis is smallerthan in the case of acute sinusitis, since the main purpose is tore-establish normal ventilation of the ethmoidal sinuses, to which endlocal-action decongestants are applied, which, accompanied by antibiotictherapy, eliminate the swelling of the mucous membrane, improve drainageand re-establish the functioning of the eustachian tube and of theethmoidal sinuses of the nose. However, the application of localdecongestants for a period of more than five days can causemedicament-induced renitis (Zeiger, R. S., Prospects for ancillarytreatment of sinusitis in the 1990s. J. Allergy Clin. Immunol., 1992,90:478). In the case of pathologies in chronic state recourse is had tosurgical intervention, cleaning the paranasal sinuses by means of directpuncturing.

In view of all that has been stated above, the treatment of acute orchronic sinusitis is a long and complicated process which, in functionof its gravity, can take several years. Furthermore, the sourcesconsulted were unable to confirm the total efficacy of the treatmentsapplied in normal practice.

The possible solution to the problems of sinusitis is to be found byseeking and studying new methods of treatment.

Known in the state of the art is the utilisation of alternativetreatments which make use of biologically active substances of naturalorigin. However, there does not exist in the state of the art anybibliography on the vast majority of substances of natural origin.

The wild plant Cyclamen europaeum L., in this invention called cyclamenplant, belongs to the Primulaceae family, and although it is a verypopular plant in many countries of the world, its use is limited todecorative purposes. Little information is available about its medicinalproperties, and there is reference only to use of the juice or powderobtained from the plant for treating headache.

On the basis of the state of the art, therefore, there exists no productas an alternative to antibiotics for treating sinusitis.

OBJECTS AND SUMMARY OF THE INVENTION

This invention resolves the disadvantages mentioned above, while it alsoprovides other advantages which will be described below.

A first objective of this invention is a method for obtaining anisolated extract of the wild plant Cyclamen europaeum L.

A second objective of this invention is the utilisation of an isolatedextract according to the invention in order to manufacture a medicamentfor treating sinusitis.

In accordance with the first objective of the invention, a method hasbeen developed for obtaining an isolated extract of the wild plantCyclamen europaeum L., which is characterised in that an aqueousextraction is carried out which comprises:

a) a first stage of pressing of the previously cleaned and preparedtuber;

b) a second stage of purification of the liquid fraction obtainedfollowing the pressing, which includes:

-   b-i) the addition of ethyl alcohol to said liquid fraction;-   b-ii) its subsequent storage in a refrigeration chamber at a    temperature between 2° C. and 25° C., preferably between 4° C. and    8° C., for 10-48 hours, preferably for 18-20 hours, and;-   b-iii) finally, filtering.

Preferably, the filtering stage b-iii) is carried out in two steps:first through a primary purification filter, and then through afine-pore bactericidal filter, at a pressure of 0.25±0.005 MPa.

Advantageously, an additional stage of pressing is carried out, for thesolid fraction of residues obtained after a first pressing stillcontains a large quantity of biologically active substances, recovery ofwhich represents an increased yield from the method on the basis ofwhich the isolated extract of the invention can be obtained. Saidadditional stage comprises:

a-i) preparing the solid fraction, residues from he pressing, by meansof addition of water in a ratio of 1:0.5-1.8, preferably 1:0.64-0.66.The value 1 refers to the weight in grams of the tuber before pressing,while the values 0.5-1.8 or 0.64-0.66 refer to the volume of water addedto the solid fraction obtained after the first pressing;

a-ii) thermostat-controlling the prepared mass to a temperature ofbetween 20° C. and 80° C., preferably between 60° C. and 70° C., for 1hour and then carrying out a second pressing; and

a-iii) finally, in a mixing reactor, mixing the liquid fraction from thesecond pressing, also called extract of residues, with the liquidfraction from the first pressing, also called juice.

From this point the procedure continues with stage b) of purification ofthe liquid fraction described above (see FIG. 1).

Optionally, a method has been developed for obtaining an isolatedextract of the wild plant Cyclamen europaeum L. which is characterisedin that an alcoholic extraction is carried out which comprises:

a) a first stage of trituration of the previously cleaned and preparedtuber;

b) a second stage of extraction of the biologically active substances,which comprises:

-   b-i) the addition of an alcohol, preferably ethanol, to the    triturate of tubers in a ratio by volume of tuber/solvent of between    1:1 and 1:5, preferably 1:3;-   b-ii) the prepared mass is brought to boiling point in alcohol and    kept there for 1 hour, before the alcoholic extraction is carried    out;

c) a third stage of purification of the alcoholic extraction, whichcomprises:

-   c-i) the addition of a sorbent in order to remove the colorants from    the alcoholic extract in a ratio of mass of raw material to mass of    sorbent of between 30:0.5 and 30:2, preferably 30:1.-   c-ii) purification in refrigerant under reflux; and-   c-iii) finally, filtering.

Advantageously, a second extraction b-iii) is carried out under the sameconditions, from which approximately 20% of the total saponins isobtained. In this case, the alcoholic extracts of the first and secondextraction are mixed in a common alcoholic extraction mixer beforeproceeding to the purification stage (see FIG. 2).

The sorbent used in the purification by alcoholic extraction is selectedfrom among activated carbon, silica gel, clay and grade II aluminiumoxide, the best results being obtained with grade II aluminium oxide.

A reference measurement of the quantity of biologically activesubstances extracted from the plant is drawn up on the basis of thehaemolytic index. The tubers of the cyclamen species contain acyclamen-saponin which hydrolyses to form cyclamyrethin sapogenin andcarbohydrates (glucoses and arabinose). Apart from saponin, sugars(glucose, fructose and other polysaccharides) have been detected,together with resinous substances, colorants and phenolic compounds. Thewater content in the tubers exceeds 70%.

The haemolytic index indices the ratio of saponins extracted. Below ahaemolytic index of 300 units there is no commercial advantage inrepeating the pressing process or obtaining a second extract, for aftertwo stages of pressing or extraction up to 96-98% of biologically activesubstances in the plant have been obtained.

After the first pressing or obtaining of the first extract, the liquidfraction has a haemolytic index of between 8,000 and 12,000 units, andafter the second pressing or obtaining of the second extract the liquidfraction has a haemolytic index of between 300 and 500 units. Additionalstages of pressing or obtaining of a third extract will not thereforepermit a liquid fraction to be obtained with haemolytic index valuessuitable from the economic point of view.

Thus, the isolated extract obtained presents a haemolytic index ofbetween 6,000 and 12,000 and an acidity value of between 5.0 and 6.8.

A second objective of this invention is the utilisation of a an isolatedextract obtained according to any of the methods described for themanufacturing of a medicament for treating sinusitis.

For this purpose, the invention proposes an isolated extract of the wildplant Cyclamen europaeum L. which includes a higher concentration ofsaponins but which also includes sugars (glucose, fructose and otherpolysaccharides), resinous substances and colorants, phenolic complexesand other additives not detected but which nevertheless confer upon theisolated extract therapeutic properties suitable for the manufacturingof a medicament for treating sinusitis.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of all that has been outlined, two figureshave been attached which, schematically and solely by way ofnon-restrictive example, show a practical case of embodiment.

FIG. 1 shows a flow diagram of the method of the invention for obtainingan isolated extract by aqueous extraction.

FIG. 2 shows a flow diagram of the method of the invention for obtainingan isolated extract by alcoholic extraction.

There follows below a more detailed description of the method forobtaining an isolated extract by means of aqueous extraction and bymeans of alcoholic extraction.

DETAILED DESCRIPTION OF THE INVENTION

There follows a description of a preferred embodiment of the method forobtaining an isloated extract of the wild plant Cyclamen Europaeum L. byaqueous extraction.

In a first embodiment of the invention, the tuber of the cyclamen plantis cleaned with running tap water until the earth, sand and otherimpurities have been completely removed from it, following which it isscrubbed with a brush in order to remove the microparticles of earth,sand, etc. The tubers are then washed with distilled water and left todry in the air. The dry tubers are then placed in a bath and coveredentirely with 95% ethanol and left for one hour.

Once clean, the tubers are placed in the press, in which the juice isexpressed mechanically. The percentage of juice or liquid fractionobtained varies between 55 and 65% in relation to the solid mass orsolid fraction after pressing.

Owing to the high haemolytic activity of the residues produced,extraction of the saponins is carried out with desalted water. Asuitable ratio of solid mass and volume of liquid needed for extractionwas determined in the course of the research to be 1:0.64-0.66.

That ratio was found to provide the greatest haemolytic activity of thesamples. The optimum temperatures for extraction are between 60 and 70°C., while the extraction time was 60 minutes. Application of saidtemperatures and time was observed to lead to maximum change of thesaponins in the aqueous extraction. The same parameters were alsooptimum from the technological point of view, given that once atemperature of 80° C. is reached the extract becomes viscous anddifficult to filter due to partial hydrolysis of the polysaccharides,while prolonging the time induces an increase in exit of the saponins.

Secondary extraction of the residues by water was found to provide ahaemolytic index of 300-500 units, suggesting a need for one extractiononly.

Once the extraction has been carried out, the mass of residues isdeposited in the press and is subjected to expression. The juice and theextract of the residues are mixed in the reactor with a mixer, providingcommon extractions therefrom.

The aqueous extractions obtained (juice of tubers+extract from theresidues), like the end products, contain major disposable impuritiesand therefore require additional cleaning.

Most of the impurities can be eliminated by adding to the extractions15% of ethyl alcohol at 96% and then keeping in the refrigerationchamber for 18-20 hours. Once that time has elapsed, the extractions arefiltered through the primary purification filter, and then the fine-porebactericidal filter. This last sterilised filtration of the resultingextractions is carried out at a pressure of 0.25±0.005 MPa.

The filtrations provide a transparent solution which remains stable fortwo months when kept in a refrigeration chamber at a temperature of 4-8°C. This solution serves as the base substance for making a medicamentfor treating sinusitis.

In a second embodiment of the invention, a method is disclosed forobtaining an isolated extract of the wild plant Cyclamen europaeum L. byalcoholic extraction.

Once clean and prepared, the tubers are cut into small pieces, placed ina jar and covered with 96% ethyl alcohol in a proportion of solid mass(raw material) and volume of solvent of 1:3, respectively. The mixtureis heated until the solvent boils and is then left for 60 minutes. Asecond extraction is then carried out under the same conditions, fromwhich an average of 20% of the total saponins is obtained.

The alcoholic extractions obtained from the first and second extractionsare combined and purified by refrigerant under reflux to eliminate thecolorants.

The purification is carried out with grade II aluminium oxide at atemperature of 40-50° C. and is repeated three times, for 15-20 minuteseach time. The optimum proportion of the mass of raw material to mass ofsorbent is 30:1, respectively.

The purified extract serves as the base substance for production of amedicament for treating sinusitis. This extract remains stable for twomonths when kept in a refrigeration chamber at a temperature of 4-8° C.

Pharmacological Results

Study of the pharmacological properties of the isolated extract of theinvention has shown that application of its light concentrates producesreflexive secretion from the nasal cavity. With said extract it ispossible to obtain solutions of different concentrations, or in otherwords to personalise the dosage.

Study of the reflexive reaction, carried out by placing 2-3 drops of amedicament prepared with the isolated extract of the invention in thenasal cavity of rabbits and dogs has shown that neither the latentreaction period nor the duration of the secretion present alterations ofany kind, irrespective of whether the preparation is administered daily(every 24 hours) or on alternate days.

The information from the histological study shows that application ofthe preparation does not cause side-effects of irritation, and stillless inflammation of the mucous membrane.

A second administration into the nasal cavity of the recommended dosesof a preparation of the isolated extract of the invention does not causeadverse alterations of the general state or behaviour of the animals.

The therapeutic action of a preparation that includes the isolatedextract of the invention is based on stimulation of the secretiveactivity of the integumentary epithelium of the mucous membrane ingeneral, together with the serum and submucous glands, which facilitatesan intense draining of the paranasal sinuses. According to histologicalinformation, the optimum period for the second intranasal administrationis 48 hours.

Concentrations of the preparation retain their efficacy in solutions ofbetween 1:100 and 1:500.

The conditions of application of the preparation, together with thecharacteristics of the reaction of administration thereof, practicallyexclude any possibility of it being absorbed from the surface of themucous membrane and producing an undesirable systemic action.

Toxicological Study

The systemic toxic properties of the isolated extract and of themedicament containing it have been studied in albino mice and rats bymeans of intraperitoneal, intravenous and oral administration, giventhat it is impossible to administer sufficiently large dosesintranasally, that is, the method recommended for the clinical trial.

The toxic manifestations in all the methods of administration weregenerally those inherent to the saponins: a generalised depression ofthe central nervous system and delayed (up to 3 days) mortality in theevent of intra-abdominal and oral administration; intravascularhaemolysis and immediate mortality in the event of intravenousadministration; localised irritant action in relation to the mucousmembranes.

The lethal single doses of oral administration, applied to mice, are:

(a) Alcoholic extract: LD50—1337.7±23 mg/kg; LD84—1894 mg/kg, LD16—781mg/kg;

(b) Aqueous extract: LD50—1466.5±37 mg/kg; LD84—2107 mg/kg, LD16—826mg/kg;

(c) Medicament: LD50—2386.84±85 mg/kg; LD84—3852 mg/kg, LD16—920 mg/kg;

According to the general toxicological classification of the LD50indices, the isolated extract and the medicament that contain it pertainto the elements of moderate or slight toxicity with localised irritantaction of moderate extent.

The single non-toxic, non-effective dose of the medicament applied tomice is 10 mg/kg, that is, 1/250 LD50, and exceeds the maximum singletherapeutic dose (0.03 mg/kg) recommended for clinical trials byapproximately 350 times.

The subtoxic toxicity of the non-toxic dose of the medicament wasstudied in mice in an experiment on therapeutic intranasal applicationcarried out over the course of two weeks, and shows the cases ofundesired penetration of the preparation from the nasal cavity into thestomach.

The second oral administration of the medicament for 14 days in a doseof 10 mg/kg was totally innocuous for the mice.

The innocuousness of the isolated extract and of the medicamentadministered five times has been shown by the results of the study ofthe specific pharmacological activity of these preparations in the eventof applying maximum “therapeutic” concentrations.

The localised irritant action of the medicament shows itself to thegreatest extent with the conjunctive tissue but was not detected in themucous membrane of the stomach, while the mucous membrane of the nasalcavity probably lies between these two as regards degree ofirritability.

The clinical trials of the medicament in solutions of 1:100 to 1:500,which is approximately equivalent to 1/350- 1/500 of the “non-toxic”oral dose applied to mice, can be considered totally innocuous,especially if account is taken of it being practically impossible forthe preparation to be absorbed or ingested from the nasal cavity.

Despite the fact that a specific embodiment of this invention has beendescribed and shown, it is obvious that an expert in the subject wouldbe able to introduce variations and modifications, or replace thedetails by others that are technically equivalent, without departingfrom the sphere of protection defined by the attached claims.

1. A method for obtaining an extract of the wild plant Cyclameneuropaeum L. for treating sinusitis, which comprises: a) cleaning atuber Cyclamen europaeum L.; b) pressing the cleaned tuber of Cyclameneuropaeum L. to obtain a first liquid fraction and a solid fraction; c)adding water to the solid fraction in ration of 1:0.5-1.8; d)thermostat-controlling the solid fraction of part (c) to a temperatureof between 20° C. and 80° C.; e) pressing the solid fraction with addedwater of part (c) to obtain a second liquid fraction; f) mixing thefirst liquid fraction with the second liquid fraction in a mixingreactor to obtain a mixture of liquid fractions; g) purifying themixture of liquid fractions of part (f) comprising: h) adding an alcoholto the mixture of part (f) and storing it in a refrigeration chamber;and i) filtering the mixture of part (f) to obtain said extract ofCyclamen europaeum L.
 2. The method as claimed in claim 1, wherein saidstorage is carried out at a temperature between 2° C. and 25° C. for10-48 hours.
 3. The method as claimed in claim 2, wherein said storageis carried out at a temperature between 4° C. and 8° C. for 18-20 hours.4. The method as claimed in claim 1, wherein said filtering is carriedout in two steps: first, through a primary purification filter and,through a fine-pore bactericidal filter, at a pressure of 0.25±0.005MPa.
 5. The method as claimed in claim 1, wherein in part (c) water isadded in a ratio of 1:0.64-0.66.
 6. The method as claimed in claim 1,wherein in part (d) thermostat-controlling the solid fraction is carriedout to a temperature from 60° C. to 70° C. for 1 hour.